Cohesive coprecipitates of sulfated polysaccharide and fibrillar protein and use thereof

ABSTRACT

The present invention concerns cohesive biopolymer gels comprising coprecipitates of sulfated polysaccharides and fibrillar proteins, exemplified by coprecipitates of dextran sulfate and gelatin, useful for clinical applications including as implants for tissue engineering as well as in biotechnology. The cohesive biopolymer gels according to the present invention may be used clinically either per se or as a scaffold for a cell-bearing implant, as a depot for sustained release of bioactive agents, or for research and biotechnology.

FIELD OF THE INVENTION

The present invention relates to compositions comprising coprecipitatesof sulfated polysaccharide and fibrillar protein, exemplified by acoprecipitate of dextran sulfate and gelatin, that form a cohesivebiopolymer having unique physicochemical attributes useful as universalbiomatrices or scaffolds for clinical applications including as implantsfor tissue engineering as well as in biotechnology. The matricesaccording to the present invention may be used clinically either per seor as a scaffold for a cell-bearing implant.

BACKGROUND OF THE INVENTION

Matrices useful for guided tissue regeneration and/or as biocompatiblesurfaces useful for tissue culture or tissue implants are well known inthe art. These matrices may therefore be considered as substrates forcell growth either in vitro or in vivo. Suitable matrices for tissuegrowth and/or regeneration and/or implants include both biodegradableand biostable entities. Among the many candidates that may serve asuseful matrices claimed to support tissue growth or regeneration, areincluded gels, foams, sheets, and numerous porous particulate structuresfabricated at different densities and in different forms and shapes.

In many instances the matrix may advantageously be composed ofbiopolymers, including polypeptides or proteins, as well as variouspolysaccharides, including proteoglycans, sulfated polyglycans and thelike. In addition, these biopolymers may be either selected ormanipulated in ways that affect their physicochemical properties. Forexample, biopolymers may be cross-linked either enzymatically,chemically or by other means, thereby providing greater or lesserdegrees of rigidity or susceptibility to degradation.

Among the manifold natural polymers which have been disclosed to beuseful for tissue engineering or culture, one can enumerate variousconstituents of the extracellular matrix including fibronectin, varioustypes of collagen, and laminin, as well as keratin, fibrin andfibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate andothers.

U.S. Pat. Nos. 5,955,438 and 4,971,954 disclose collagen-based matricescross-linked by sugars, useful for tissue regeneration.

U.S. Pat. No. 5,948,429 disclosing methods of making and usingbiopolymer foams comprising extracellular matrix particulates.

U.S. Pat. Nos. 6,083,383 and 5,411,885 disclose fibrin or fibrinogenglue and methods for using same. U.S. Pat. Nos. 5,279,825 and 5,173,295disclose a method of enhancing the regeneration of injured nerves andadhesive pharmaceutical formulations comprising fibrin. U.S. Pat. No.4,642,120 discloses the use of fibrin or fibrinogen glue in promotingrepair of defects of cartilage and bone.

U.S. Pat. Nos. 6,124,265 and 6,110,487 disclose methods of making andcross-linking keratin-based films and sheets and of making porouskeratin scaffolds and products of same.

Hyaluronic acid (HA) is a naturally occurring high molecular weightlinear polymer belonging to the glycosaminoglycan family, composed ofrepeating units of glucuronic acid and N-acetyl glucosamine. HA readilyforms hydrated gels which serve in vivo as space filling substance. Theutility of hyaluronic acid as a beneficial component for supportingtissue growth is well established in the art, as exemplified in U.S.Pat. No. 5,942,499, which discloses methods of promoting bone growthwith hyaluronic acid and growth factors. U.S. Pat. Nos. 5,128,326 and5,783,691 disclose methods of producing and using cross-linkedhyaluronans in promoting tissue repair and as reservoirs for bioactiveagents including drugs or growth factors.

Laminin (LN) is an adhesive glycoprotein of high molecular weight, whichis known as a major cell matrix binding component. U.S. Pat. Nos.4,829,000 and 5,158,874 exemplify uses of gels or matrices comprisinglaminin.

WO 92/21354 discloses biocompatible anionic polymers that inhibitfibrosis, scar formation and surgical adhesions. Anionic polymers foruse in the invention include but are not limited to naturalproteoglycans, and the glycosaminoglycan (GAG) moieties ofproteoglycans. The anionic polymers dextran sulfate and pentosanpolysulfate are preferred, and according to a more preferred embodimentDextran Sulfate, preferably with a molecular weight of 40,000 to 500,000Daltons in which the sulfur content is greater than about 10% by weightis used.

U.S. Pat. No. 5,861,382 and U.S. Pat. No. 6,020,323 disclose substancescomprising carboxylated or sulfated oligo-saccharides in substantiallypure form, and methods of using same for the regulation of cytokineactivity in a host.

One of the present inventors has previously disclosed (WO 01/02030) adevice with a constant perfusion system for maintenance of viable cells,tissues and composite implants. That disclosure further concerns ascaffold which is used as a growth supportive base for various cells andtissue explants comprising naturally derived connective tissue orskeletal tissue, cross-linked with one of the following: HA,proteoglycans, GAGs, chondroitin sulfates, heparan sulfates, heparinsand dextran sulfates.

Cross-linking between macromolecules of the extra-cellular matrix mayoccur naturally (Laurent, 1964; Wang and Bozos, 1985). In vitro, heparinwas reported to interact with natural occurring mammalian's proteinssuch as amyloid (Cohlbery et. al., 2002) or S-100 (Robinson et. al.,2002).

Certain specific combinations of polysaccharides and fibrillar proteinshave been used to promote cell growth. For example, the non-sulfatedpolysaccharide chitosan has been combined with gelatin as a scaffoldsupporting chondrocyte growth and differentiation in vitro (Risbud M. etal. 2001); vascular cells responded in vitro and in vivo to chitosan anddextran sulfate (Chupa et.al., 2000); and auricular chondrocytes ofelastic cartilage were shown to grow in hydrogels of collagen andalginate, a non-sulfated polysaccharide composed of polymannuronic acid(de-Chalain et. al., 1999).

U.S. Pat. No. 4,280,954 discloses composite materials which are formedby contacting collagen with a mucopolysaccharide and subsequentlycovalently cross-linking the resultant polymer. U.S. Pat. No. 4,448,718discloses that the cross-linking is performed by gaseous aldehyde, andU.S. Pat. No. 4,350,629 further discloses that if collagen fibrils arecontacted with a cross-linking agent before being contacted with theglycosaminoglycan, the materials produced have extremely low levels ofthrombogenicity

U.S. Pat. Nos. 5,866,165 and 5,972,385 disclose a method for preparing amatrix, the method comprising reacting a polysaccharide with anoxidizing agent to open sugar rings on the polysaccharide to formaldehyde groups, and reacting the resulted aldehyde groups to formcovalent linkages to collagen, and the use of the matrix to support thegrowth of tissue, such as bone, cartilage or soft tissue. U.S. Pat. No.6,309,670 discloses the use of this matrix, which further comprises adifferentiation factor for the treatment of a bone tumor.

U.S. Pat. No. 6,624,245 discloses a composition prepared by admixture ofindividually reactive polymer components, wherein the admixtureinitiates rapid cross-linking and gel formation, wherein suchcompositions are suited for use in applications in which rapid adhesionto the tissue and gel formation is desired, including using thecompositions as bioadhesives, for tissue augmentation, in the preventionof surgical adhesions, for coating surfaces of synthetic implants andthe like.

Dextran sulfate alone was found to act as an antimicrobial agent(Christensen et al., 2001) and as prophylaxis for peritoneal cancermetastasis (Hagiwara et al., 2000). It was also used as an antifoamagent of proteins (Ibanoglu et. al., 2001)

Nowhere in the background art is it taught or suggested that biopolymerscomprising fibrillar proteins coprecipitated with sulfatedpolysaccharides in general and dextran sulfate in particular, have novelphysicochemical properties. Furthermore, the use of these scaffoldmatrices as an implant suitable for transplantation, per se or as cellbearing implants, has never been disclosed.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a cohesivebiopolymer gel that is biocompatible and affords a convenientenvironment for tissue repair. It is a further object of the presentinvention to provide a universal biopolymer scaffold suitable for manycell bearing implants which may conveniently be used either in vitro orin vivo. It is a further object of the present invention to provide ascaffold or gel which is useful for clinical applications due to itsunique attributes of fostering tissue regeneration.

The cohesive biopolymer may be fabricated in the form of a gel, sleeve,cuff, sponge, membrane or any other shape useful as a scaffold fortissue repair.

These and other objects of the present invention are met by cohesivebiopolymer gels comprising a coprecipitate of at least one sulfatedpolysaccharide and at least one fibrillar protein, wherein thecoprecipitate is formed in the absence of an exogenous cross-linkingagent in the presence of a volatile organic solvent. Surprisingly, thecoprecipitation of a fibrillar protein with a polyanionic polysaccharideprovides a gel with highly advantageous properties for use in vivo.According to some embodiments the polyanionic sulfated polysaccharide isselected from the group consisting of dextran sulfate, chondroitinsulfate, heparan sulfate, heparin, keratan sulfate, dermatan sulfate, aswell as algal polyglycan sulfates, or synthetic sulfatedpolysaccharides, as are known in the art. According to some embodimentsthe fibrillar protein is selected from the group consisting of collagen,elastin, fibrin, albumin and gelatin.

According to one currently more preferred embodiment exemplified herein,dextran sulfate is coprecipitated with gelatin.

Though it is possible to use gelatin obtained from human collagen, morepreferred are materials of non-human origin, due to safety concernsrelated to the use of collagens obtained from human sources. Accordingto certain embodiments porcine or bovine gelatin are used to form thecoprecipitates of the invention, though other mammalian species may alsobe used.

Any type of dextran sulfate may be employed according to the principlesof the present invention, providing different biopolymer propertiesaccording to the molecular weight of the dextran sulfate used. Accordingto one embodiment of the present invention the dextran sulfate has amolecular weight of from about 4,000 Dalton to about 500,000 Dalton.

According to some embodiments, dextran sulfate having high molecularweights are preferred, particularly dextran sulfate having a molecularweight of more than 300,000 Daltons, preferably more than 400,000Dalton, most preferably dextran sulfate of about 500,000 Dalton.

According to alternative embodiments, dextran sulfate having lowmolecular weights are preferred, particularly dextran sulfate having amolecular weight below 10,000 Daltons, preferably below 8,000 Dalton,most preferably dextran sulfate of about 5,000 Dalton.

The properties of the cohesive biopolymer gel of the present inventionare dependent on the conditions under which the dextran sulfate and thegelatin coprecipitate, particularly on the pH during thecoprecipitation. According to one embodiment the coprecipitate is formedat a pH of at least 2 pH units above or below neutral pH.

According to some embodiments, high molecular weight dextran sulfate(i.e. of more than 300,000 Dalton) is coprecipitated with gelatin underacidic pH conditions. According to one preferred embodiment, the acidicpH conditions comprise a pH in the range of from about 2.0 to about 5.0,more preferably from about 2.0 to about 4.0.

According to alternative embodiments, low molecular weight dextransulfate (i.e. dextran sulfate of bellow 10,000 Dalton) is coprecipitatedwith gelatin under basic pH conditions. According to one preferredembodiment, the basic pH conditions comprise a pH in the range of fromabout 8.0 to about 12.0, preferably from about 9.0 to about 12.0, mostpreferably from about 9.0 to about 11.0.

The acidic and basic pH conditions enable the coprecipitation of thefilbrillar protein and the sulfated polysaccharide. The coprecipitationis further enhanced in the presence of a volatile, preferably non-toxicorganic solvent. According to one embodiment, the volatile organicsolvent is an alcohol.

According to one embodiment, the present invention provides a method forpreparing the biocompatible cohesive biopolymer gel of the presentinvention comprising:

providing a solution of a fibrillar protein;

providing a solution of a sulfated polysaccharide;

combining the two solutions at appropriate pH in the absence ofexogenous cross-linking agent to form a coprecipitate of cohesivebiopolymer; and

precipitating the cohesive biopolymer with a volatile organic solvent.

According to preferred embodiments the coprecipitates may be formed ormolded to any desired shape. According to certain embodiments the gelsmay be dried and stored prior to use. In these embodiments the gels arerehydrated with a suitable medium prior to use.

In order to provide the cohesive biopolymer coprecipitate with desiredattributes, e.g. tensile strength, surface charge, density, porosity,ability to withstand suturing without tearing, etc., it is possible toadd optional components either as a separate layer or interspersed ordispersed within the novel biopolymer of the invention.

It should be understood that the interaction between the sulfatedpolysaccharides and fibrillar protein resulting in the cohesivebiopolymer coprecipitate of the invention may be covalent, non-covalent,or electrostatic. Cross-linked copolymers may provide furtherimprovements to the product. According to optional features of theinvention it may be advantageous to utilize cross-linking agents toalter or stabilize the attributes of the sulfated polysaccharidefibrillar protein coprecipitate. Cross-linking agents are known in theart and may include: simple sugars including pentoses or hexoses;aldehydes including glutaraldehyde; or synthetic cross-linkers ifappropriate. According to one currently preferred embodiment, the crosslinking agent is ribose.

According to a first aspect of the current invention we disclose aninnovative material made of gelatin and dextran sulfate, having uniqueadvantageous properties. The new cohesive biopolymer allows thepreparation of articles of various shapes, including but not limited totubes and sheets, and any other desired shape or form. When the shapedarticles are not used immediately, they may be dehydrated for storage,and then re-hydrated prior to use.

As exemplified herein the novel coprecipitates disclosed according tothe present invention are suitable for many clinical applications,including as a support or as a guide for peripheral nerve regeneration,as a sleeve for coating or enclosing the spinal cord, as a patch forrepairing a lesion in a tissue, as a membrane for repairing tracheallesions, as a coating or envelope for a vascular or tracheal stent.

The novel biopolymers of the invention are useful in the fabrication ofmedical devices, the form or shape of these devices depending on thespecific intended use. The methods for fabrication of these devices mayvary widely depending on the intended use.

These biopolymers are suited for use as fibers which fibers can befabricated by conventional processes such as dry extrusion, gelextrusion, melt extrusion, solution extrusion or spinning extrusion,spraying of nanofibrils with or without an electromagnetic field, or bycombination of these processes. The fibers can then be dried and spooledonto spools. The fibers can be woven, knitted, bundled or braided intocomplex form or constructs by methods known from industrial applicationsof textile manufacture.

The degree of solubility of the biopolymer matrices according to thepresent invention in various aqueous or organic solvents depends on thespecific sulfated polysaccharide used to interact with the protein ofchoice, and on the coprecipitation conditions. According to someembodiments, the biopolymer of the invention disintegrates intometabolic degradable substances, which are soluble in aqueous solvents.

The biopolymer of the present invention is suited for extrusion andco-extrusion with different components, organic or inorganic in natureand polymeric or otherwise, including multiple components, multilayeredtypes of fiber as well as hollow fibers and tubes.

According to one currently more preferred embodiment, dextran sulfate iscoprecipitated with gelatin, to provide cohesive biopolymer gel withunexpectedly advantageous chemical and physical properties, in additionto its biological properties of biocompatibility, controllablebiodegradation rate, affinity for cultured cells, and fostering cellgrowth. The novel cohesive biopolymer has physicochemical propertiesdifferent from those of the uncombined raw materials, as can beevaluated by MRI analyses, infrared spectrum, elution from gelseparation columns and other analytical tools known in the art.

According to a first embodiment of the invention, these biopolymers areuseful per se as a biocompatible implant for guided tissue regenerationor tissue engineering. According to a further embodiment of the presentinvention these biopolymers are useful when they further compriseimplants bearing cells to be transplanted to a site of injury or toameliorate tissue impairment. According to a further embodiment of thepresent invention the cohesive biopolymer gels further compriseadditional bioactive molecules to enhance tissue repair or regeneration.

Methods of using these cohesive biopolymers in vivo in clinicalapplications are disclosed, whereby the cohesive biopolymer gelsaccording to the present invention may be used clinically either per seor as a scaffold for a cell bearing implant, alone or with additionallayers of components. The cohesive biopolymers according to the presentinvention may advantageously be used as a substrate suitable forsupporting cell selection, cell growth, cell propagation anddifferentiation in vitro as well as in vivo.

The cohesive biopolymers according to the invention comprise dextransulfate in the range of about 30% to about 70% (w/w) and gelatin in therange of about 30% to 70% (w/w). This range of ingredients providesscaffold with the desired properties in terms of flexibility andelasticity. Typically, the biopolymer of the invention may convenientlybe formed by interaction of approximately equal amounts of dextransulfate and gelatin. According to one embodiment, the biopolymer of theinvention is formed by interaction of 70% gelatin with 30% dextransulfate.

The present invention also provides for the addition of ther activeingredients to the biopolymers comprising dextran sulfate and gelatin,including but not limited to other proteins such as fibrin, albumin,collagen, elastin and lysozyme; one of the diverse polysaccharidesproteoglycans and hyaluronate; cross-linkers such as factor XIII,lysyloxidase; anticoagulants; growth factors; antioxidants and the like.These optional additives may be incorporated in such a manner to providefor desired pharmacokinetic profiles. Within the scope of the presentinvention there are provided methods of using the dextransulfate-gelatin biopolymer gels for sustained release of bioactivecomponents in vivo. In other instances the additives may be incorporatedin such a manner to provide for short-lived optimal local concentrationsof the bioactive molecules incorporated therein.

The physicochemical parameters of the cohesive biopolymer gel mayreadily be optimized in accordance with the intended use of thescaffold, and methods are disclosed to provide guidance to the skilledartisan in optimization.

The present invention is explained in greater detail in the description,figures and claims below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, 2, 3 Different views of a nerve cuff made from a biopolymer ofdextran sulfate combined with gelatin (GD biopolymer) according to theinvention.

FIG. 4 Gel filtration profiles on Sepharose CL-6B column of a) dextransulfate vs. coprecipitated biopolymer of gelatin and dextran sulfate (GDbiopolymer) b) gelatin vs. GD biopolymer.

FIG. 5 Nuclear Magnetic Resonance spectra of a) dextran sulfate; b)gelatin; and c) coprecipitated biopolymer of gelatin and dextransulfate.

FIG. 6 Infrared spectra of a) dextran sulfate; b) gelatin; and c)combined biopolymer of gelatin and dextran sulfate.

FIG. 7 Swelling degree of GD membranes NVR-3 in distilled water beforeand after cross-linking.

FIG. 8 Swelling degree of GD membranes NVR-3 in simulated salivasolution before and after cross-linking.

FIG. 9 Degradation rate of GD-membranes NVR-3 cross-linked with riboseover time.

FIG. 10 Porosity of dry GD biopolymer membrane, as shown by scanningelectron microscopy.

FIG. 11 Embryonic rat spinal cord cells on NVR-7 tube after 45 days ofgrowth. (magnification: ×100).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Sulfated Polysaccharide-Protein Coprecipitates

The present invention provides a biocompatible scaffold gel comprising anew polymeric material. The biopolymer of the present invention hasunique chemical and mechanical properties as well as cell growthpermissive features, and is therefore suitable for use as an implant,with or without cells.

Various gels or matrices generated from proteins in conjunction withother biocompatible polymers have shown great promise in the area oftissue engineering. According to the present invention we now disclosenovel coprecipitates comprising at least one fibrillar protein and atleast one sulfated polysaccharide. These novel coprecipitates areobtained in the absence of an exogenous cross linking agent, in thepresence of a volatile organic solvent. The coprecipitates so obtainedare subsequently shaped into any desired shape or geometry as requiredfor a particular application. They may be further cross linked with anadditional cross linking agent after the initial coprecipitate hasformed. According to various embodiments various additives may beadvantageously added to the gels prior to their formation or prior touse.

According to one exemplary embodiment the coprecipitates of the presentinvention comprise gelatin and dextran sulfate. Gelatin-Dextran sulfate(G-D) coprecipitates are useful for guiding tissue regeneration and ascell carriers in various clinical applications or other fields.

The polymers composing the cohesive biopolymer gel of the presentinvention were selected to simulate the two main constituents of thematrices of common connective tissues, namely collagens andglycosaminoglycans. The cohesive biopolymers gel of the presentinvention comprises a coprecipitate of sulfated polysaccharides andgelatin or other fibrillar proteins. According to one embodiment,sulfated polysaccharides include dextran sulfate, chondroitin sulfate,heparan sulfate, heparin, keratan sulfate, dermatan sulfate, as well asalgal polyglycan sulfates, or synthetic sulfated polysaccharides as areknown in the art. According to one currently preferred embodimentgelatin is used to mimic the collagen, and dextran sulfate is used tosimulate the glycosaminoglycans in forming the biopolymer gel of thepresent invention. The term “gel” is used herein in the conventionalsense to refer to water-swellable polymeric matrices that can absorb asubstantial amount of water to form elastic gels. The gel may behydrated to obtain a dry gel. Upon placement in an aqueous environment,dry gels swell to the extent allowed by the degree of the interactionsbetween the gel-forming polymers.

Dextran is a glucose polymer and dextran sulfate is a polysaccharide,composed of sulfated glucose as the repeating units. It contains about17%-20% sulfur with up to three sulfated groups per glucose molecule.The molecular weight of dextran sulfate is in the range of 4,000 to500,000 KD. As used herein, the term “molecular weight” refers to theweight of average molecular weight of a number of molecules in any givensample, as commonly used in the art. Thus, a sample of Dextran sulfate5,000 kD might contain a statistical mixture of polymer moleculesranging in weight from, for example, 4,500 to 5,500 kD, with onemolecule differing slightly from the next over a range. Variations inthe dextran sulfate molecular weight are associated with differences inits biological activity.

Use of sulfated polysaccharides of various molecular weights andapplying different coprecipitation conditions in producing thegelatin-sulfated polysaccharides biopolymer of the present inventionresult in a gel having different properties.

Use of a high molecular weight dextran sulfate in the production of GDmatrix according to the present invention results in a readilybiodegradable matrix. Use of low molecular weight dextran sulfateprolongs the retention time of the biopolymer gel in vitro and in vivo,and results in a biopolymer with increased strength and elasticity.

According to one embodiment of the present invention the dextran sulfatehas a molecular weight of from about 4,000 Dalton to about 500,000Dalton. According to another embodiment, the GD biopolymer of thepresent invention is produced with a high molecular weight dextransulfate having a molecular weight of more than 300,000 Dalton,preferably more than 400,000 Dalton, most preferably with dextransulfate having a molecular weight of about 500,000 Dalton.

According to alternative embodiment, the GD bioploymer of the presentinvention is produced with dextran sulfate of low molecular weighthaving a molecular weight of below 10,000 Dalton, preferably below8,000, most preferably dextran sulfate having a molecular weight ofabout 5,000 Dalton.

According to the present invention dextran sulfate of any source may beused, including dextran sulfate commercially available, syntheticallyprepared, or isolated from natural source. According to one embodiment,the dextran sulfate used according to the present invention is ofbacterial origin, which best simulates the glycosaminoglycans.

Gelatin is a heterogeneous mixture of water-soluble proteins of highaverage molecular weight. Gelatin is not found in nature but is derivedform collagen by hydrolytic action. Gelatin is obtained by boiling skin,tendons, ligaments, bones etc., with water. Approximate amino acidcontent of gelatin is: glycine—25.5%, alanine 8.7%, valine 2.5%, leucine3.2%, isoleucine 1.4%, cystine and cysteine 0.1%, methionine 1.0%,phenylalanine 2.2%, threonine 1.9%, tyrosine 0.5% aspartic acid 6.6%glutamic acid 11.4%, arginine 8.1% lysine 4.1% and histidine 0.8%. Thetotal is over 100% because water is incorporated into the molecules ofindividual acids.

Though it is possible, according to the present invention, to usegelatin obtained from human collagen, more preferred are materials ofnon-human origin, due to safety concerns related to the use of collagenobtained from human sources. According to one embodiment, the gelatin isof mammalian species other than human. According to one currentlypreferred embodiment the gelatin to be used is porcine or bovinegelatin.

The coprecipitation of dextran sulfate and gelatin is generallyperformed in a controlled manner, i.e. using a specific ratio of dextransulfate to gelatin, dextran sulfate of certain molecular weight,specific pH conditions and certain incubation time, thus controlling thedegree of interaction between to two polymers. The coprecipitate isformed in the absence of an exogenous cross-linking agent in thepresence of a volatile organic solvent. The resulted GD cohesivebiopolymer of the invention comprises dextan sulfate in the range offrom about 30% to about 70% (w/w) and gelatin in the range of about from30% to about 70% (w/w). This range of ingredients provides scaffold withthe desired properties in terms of flexibility, elasticity andbiodegradability. According to one embodiment, the biopolymer of theinvention is formed by coprecipitating approximately equal amounts ofdextran sulfate and gelatin. According to another embodiment, thebiopolymer of the invention is formed by interaction of 70% gelatin with30% dextran sulfate. The interaction between the dextran sulfate andgelatin resulting in the coprecipitated biopolymer of the presentinvention rnay be covalent, non-covalent or electrostatic.

The properties of the cohesive biopolymer gel of the present inventionalso depend on the conditions under which the coprecipitation of dextransulfate and gelatin is performed, particularly the pH range of theinteraction medium.

According to one embodiment, the coprecipitate is formed at a pH of atleast 2 pH units above or below natural pH. According to one embodiment,the coprecipitation of gelatin and dextran sulfate of high molecularweight is performed under acidic pH conditions. According to onecurrently preferred embodiment, the coprecipitation of gelatin anddextran sulfate having molecular weight of above 300,000 Dalton isperformed at a pH range of from about 2.0 to about 5.0, preferably fromabout 2.0 to about 4.0.

According to alternative embodiment, the coprecipitation of gelatin anddextran sulfate of low molecular weight is performed under basic pHconditions. According to one embodiment, the coprecipitation of gelatinand dextran sulfate having a molecular weight below 10,000 Dalton isperformed at a pH range of from about 8.0 to about 12.0, preferably fromabout 9.0 to about 12.0, most preferably from about 9.0 to about 11.0.

The novel cohesive biopolymer gel of the present invention is obtainedby the coprecipitation of gelatin and dextran sulfate from the aqueoussolution under extreme pH conditions, either acidic or basic. To furtherenhance this spontaneous coprecipitation, a volatile organic solvent maybe added to the solution. According to one embodiment, the volatileorganic solvent is an alcohol, particularly ethanol. The resultedcoprecipitate is removed from the solution and it may be then firstshaped or directly dehydrated. The matrix may be dried at ambient airtemperature or at elevated temperature. During the dehydration processthe organic solvent evaporates; thus, no chemical reagents that mightimpair the bioavailability of the resulting gel are present in the finalproduct. According to one embodiment, the volatile organic solvent isnon-toxic, and preferably is an alcohol. The dehydrated matrix is readyfor storage, and should be re-hydrated prior to use.

According to one embodiment, the present invention provides a method forpreparing the biocompatible cohesive biopolymer gel of the presentinvention comprising:

providing a solution of a fibrillar protein;

providing a solution of sulfated polysaccharide;

combining the two solutions at appropriate pH in the absence ofexogenous cross-linking agent to form a coprecipitate of cohesivebiopolymer; and

precipitating the cohesive biopolymer with a volatile organic solvent.

In order to provide the coprecipitated matrix with other desiredattributes, e.g. tensile strength, surface charge, density, porosity,ability to withstand suturing without tearing, etc., it is possible toadd optional components either as a separate layer or interspersed ordispersed within the novel biopolymer of the invention.

According to one embodiment, the gelatin-dextran sulfate matrix of thepresent invention can also be further cross-linked to alter or stabilizethe attributes of the GD polymer by means of any of a number ofconventional chemical cross-linking agents, including, but not limitedto simple sugars including pentoses or hexoses, glutaraldehyde, divinylsulfone, epoxides, carbodiimides, and imidazole. The concentration ofchemical cross-linking agent required is dependent on the specific agentbeing used and the degree of cross-linking desired. The cross linkedpolymer may offer increased reproducibility as well as otherimprovements to the product, including increased retention time in vitroand in vivo and increased strength. According to one currently preferredembodiment, the cross-linking agent is ribose.

The novel cohesive biopolymer of the present invention hasphysicochemical properties different from those of the uncombined rawmaterial. As exemplified herein below, characterization of the GDbiopolymer by gel filtration chromatography, nuclear magnetic resonancespectroscopy (NMR) and infrared spectroscopy shows that the GDcoprecipitate cohesive biopolymer according to the present invention isclearly distinguished from gelatin or dextran sulfate alone.

The GD cohesive biopolymer is easily sterilized and stored at roomtemperature, capable of large-scale production and moldable into variousshapes, including but not limited to tubes and sheets suitable for thesupport of a guide for peripheral nerve regeneration, a sleeve forcoating or enclosing the spinal cord, a patch for repairing a hole in atissue, particularly closing tracheal holes, and a coating or envelopefor a vascular and tracheal stent. The biopolymer is useful in thefabrication of medical devices, the form or shape of these devicesdepending on their intended use. The method for fabrication of thesedevices may vary widely depending on the intended use. The ability tocontrol the physicochemical properties of the GD biopolymer according tothe present invention allow for the broad range of shapes and utilitiesof this scaffold biopolymer gel. The GD-biopolymer could be designed tobe more rigid or more flexible; readily biodegradable or with elongatedretention time; insoluble in water or with moderate solubility, and thelike. As exemplified herein below, the GD biopolymer was shaped into atube, that designed to help restore function to patients with peripheralnerve injures (whole sectional loss) by acting as a bridge for guidingthe nerve regeneration (also see FIGS. 1, 2, 3). A shape of a membranewas also designed, and the membrane was used to cover a hole in a rabbittrachea. The membrane was sewed onto the trachea, and the specific GDbiopolymer type used enabled the sewing without rupturing the membrane.

The biopolymer is suited for use as fibers which fibers can befabricated by conventional processes such as dry extrusion, gelextrusion, melt extrusion, solution extrusion or spinning extrusion orby combination of these processes. The fibers can then be dried andspooled onto spools. The fibers can be woven, knitted, bundled orbraided into complex form or constructs by methods known from industrialapplications of textile manufacture.

The biopolymer is suited for extrusion and co-extrusion with differentcomponents, organic or inorganic in nature and polymeric or otherwise,including multiple components, multilayered types of fiber as well ashollow fibers and tubes.

By using suitable methods as are known in the art it is possible tooptimize this material for biocompatibility, cytotoxicity aspects, andother desired parameters including rate of biodegradability, tensilestrength of fibers, flexibility of sheets and tubes, porosity, etc.

For controlling the biodegradation rate of the cohesive biopolymerproducts (i.e., to increase or decrease their biodegradability) it ispossible to add a polymerizable macromolecule with knownbiocompatibility and known degradation time, exemplified but not limitedto collagen, polyurethane, polyglycolic/polylactic acids, trimethylenecarbonate, among others. The improved material with for exampleincorporated carbonate and/or dioxanone linkages are selected to improvevarious properties of the material, particularly increasing viscosity,viscoelasticity and retention time, while prolonging yet preservingbiodegradability.

Furthermore, we provide methods for allowing the presence of poreswithin the biopolymer material and for determining the preferred poresizes. Pores may be desirable in relation to stimulation of celladhesion, growth, and differentiation, and in the converse intactnessmay be needed for certain applications such as for formation of atracheal stent.

According to one embodiment, the matrix of the present inventionfabricated in either of the shapes described above, is useful per se asa biocompatible implant for use in a variety of medical applications,including, but not limited to vascular grafts, artificial organs, heartvalves and for guided tissue regeneration or tissue engineering.

According to another embodiment these matrices are useful when theyfurther comprise implant bearing cells to be transplanted to a site ofinjury or to ameliorate tissue impairment. The cohesive biopolymersaccording to the present invention may advantageously be used as asubstrate suitable for supporting cell selection, cell growth, cellpropagation and differentiation in vitro as well as in vivo.

According to a further embodiment of the present invention the matricesfurther comprise additional bioactive molecules to enhance tissue repairor regeneration. As used herein, bioactive molecules describe moleculesexemplified by, but not limited to growth factors, cytokines and activepeptides (which may be either naturally occurring or synthetic), whichaid in the healing or re-growth of normal tissue. The function of suchbioactive molecules include stimulating local cells to produce newtissue and/or attracting cells to the cite in need of correction.Biologically active molecules useful in conjunction with the cohesivepolymer of the present invention include, but are not limited to,cytokines: interferons (IFN), tumor necrosis factors (TNF),interleukins, colony stimulating factors (CSFs); growth factors:osteogenic factor extract (OFE), epidermal growth factor (EGF),transforming growth factor (TGF) alpha, TGF-β (including any combinationof TGF-β), TGF-β1, TGF-β2, platelet derived growth factor (PDGF-AA,PDGF-AB, PDGF-BB), acidic fibroblast growth factor (FGF), basic FGF,connective tissue activating peptides (CTAP), β-thromboglobulin,insulin-like growth factors, erythropoietin (EPO), and nerve growthfactor (NGF); proteins: fibrin, albumin, collagen, elastin and lysozyme;The term “bioactive molecules” as used herein is further intended toencompass drugs such as antibiotics, anti-inflammatories,antithrombotics, and the like; one of the diverse polysaccharidesproteoglycans and hyaluronate; cross-linkers such as factor XIII,lysyloxidase; anticoagulants; and antioxidants. These optional additivesmay be incorporated in such a manner to provide for desiredpharmacokinetic profiles. Within the scope of the present inventionthere are provided methods of using the dextran sulfate-gelatinbiopolymer gels for sustained release of bioactive components in vivo.In other instances the additives may be incorporated in such a manner toprovide for short-lived optimal local concentrations of the bioactivemolecules incorporated therein.

The following examples are intended to illustrate the principles of theinvention and are to be construed in a non-limitative manner.

EXAMPLES Example 1 Manufacture of the GD Biopolymer

A. Gelatin-High Molecular Weight Dextran Sulfate Biopolymer, AcidicpH—NVR-3

A solution of Gelatin (20 mg/ml in Hanks Balanced Salt Solution (HBSS)from Gibco, Catalog #14025-50) and a solution of dextran sulfate (M.W.500,000 Dalton, 10 mg/ml in HBSS) were mixed at 70° C. in the proportionof 70% of gelatin to 30% of dextran sulfate by weight (w/w), so that thefinal concentrations are 20 mg/ml of gelatin and 10 mg/ml of dextransulfate. The ratio of 70/30 (w/w) of gelatin to dextran sulfate wasfound optimal for this combination of gelatin-dextran sulfate. After 3min., the pH of the solution was adjusted to 3.0 using 5N acetic acid(0.1 ml of acetic acid solution per 1 ml of mixture), and the solutionwas mixed carefully by shaking. After additional 3 min. a coprecipitategel was formed and it was further precipitated from the solution withabsolute ethanol. The unpolymerized molecules remain soluble, while thecohesive biopolymer is removed from liquid as a precipitate. Theprecipitated gel was collected and removed from the solution by aspatula. The collected gel-like matrix was air-dried at roomtemperature, until the matrix reached a constant weight. The drygel-like material was immersed in a 1% ribose in 80% ethanol solutionand incubated at room temperature for 7 days for further cross-linking.Alternatively, the biopolymer can be cross-linked by thermal treatmentor by other chemical agents, for example, acetone, ethyl-3(3-dimethylamino) propyl carbodiimide (EDC) oxidizing agents that are capable offorming active groups like aldehydes. For example sodium periodate iscapable of forming aldehydes readily reactive with free amino groupfollowed by reduction with sodium borohydride. The final polymer has ahigh viscosity, almost semisolid, with a high wet tensile strengtharound 70-75 MPa and high resistance to mechanical cutting (e.g., by asurgical suture of 20N).

B. Gelatin-Low Molecular Weight Dextran Sulfate Biopolymer, AcidicpH—NVR-5

A solution of gelatin (20 mg/ml in HBSS) and a solution of dextransulfate (M.W. 5,000 Daltons, 20 mg/ml in HBSS) were mixed at 70° C. inthe proportion of 50/50 by weight, so that the final concentrations were5 mg/ml of gelatin and 5 mg/ml of dextran sulfate. The mixture wasincubated for 3 min. The pH was then adjusted to pH 3.0 using 5N aceticacid (0.1 ml of acetic acid solution per 1 ml of the polymeric mixture).The dispersion was mixed carefully by shaking for additional 3 min. Theformed coprecipitate gel was further precipitated with absolute ethanoland removed by a spatula. The polymeric gel was dried under ambienttemperature until a constant weight for reached. The dry biopolymer gelwas further incubated in 1% ribose solution in 80% absolute ethanol for7 days for the formation of additional cross-linking.

The traces of acidity remained after the process of preparation of NVR-3and NVR-5 interferes with the compatibility of the gel as acells-bearing matrix. The resulted gel was relatively soluble in aqueoussolution, and therefore readily degradable. NVR-6 and NVR-7 weretherefore produced, with low molecular weight dextran sulfate.

C. Gelatin-High Molecular Weight Dextran Sulfate Biopolymer, BasicpH—NVR-6

A solution of Gelatin (20 mg/ml HBSS) and a solution of dextran sulfate(M.W. 500,000 Dalton, 20 mg/ml in HBSS) were mixed in the proportion of50% of gelatin to 50% of dextran sulfate by weight (w/w), so that thefinal concentrations are 10 mg/ml of gelatin and 10 mg/ml of dextransulfate. The pH was adjusted to 11.0 using 10N ammonium hydroxide(0.0125 ml ammonium hydroxide per 1 ml of the mixture). The mixture wasagitated at 120-150 rpm for 24 h at room temperature. The formedcoprecipitate gel was further precipitated with absolute ethanol, andthan removed from the solution by a spatula The resulted gel was driedin ambient temperature, and was found to be insoluble in aqueoussolutions.

D. Gelatin-Low Molecular Weight Dextran Sulfate Biopolymer—NVR-7

A solution of gelatin (20 mg/ml in HBSS) and a solution of dextransulfate (M.W. 5,000 Daltons, 20 mg/ml in HBSS) were mixed in theproportion of 50/50 by weight, so that the final concentrations were 10mg/ml of gelatin and 10 mg/ml of dextran sulfate. The pH was adjusted to11.0 using 10N ammonium hydroxide (0.0125 ml ammonium hydroxide per 1 mlof the mixture). Alternatively, another bases may be used, for examplediisopropylenamine. The mixture was agitated at 120-150 rpm for 24 h atroom temperature. The formed coprecipitate gel was further precipitatedwith absolute ethanol, and than removed from the solution by a spatula.Typically, the resulted NVR-7 matrix showed high strength, and thereforean additional cross-linking was not applied. However, if a strongermatrix is desired, cross-linking can be performed as described above.

All the NVR products described above were found to be biocompatible bothin vitro and in vivo, and are therefore useful in the fabrication ofmedical devices, the form or shape of these devices depending on theintended use, and the method for fabrication also depending on the usemay vary widely. The NVR-3 and NVR-5 biopolymers were found to be lesssuitable for cell growth, specifically neuronal cell growth, probablydue to a high density of negative SO₄ ⁻² charge, while NVR-6 and NVR-7were found to be highly suitable for cell growth. For example, embryonicrat spinal cord cells were found to grow successfully and sprout on thesurface of the construct (FIG. 11). The NVR-7 matrix, shaped as amembrane was found to be intact after 45 days of cell growth. Nointerference to the cell growth by the membrane was observed.

The biopolymers are also suited for use as fibers, which can befabricated by conventional processes such as dry extrusion, gelextrusion, melt extrusion, solution extrusion or spinning extrusion orby combination of these processes. The fibers can then be dried andspooled onto spools. The fibers can be woven, knitted, bundled orbraided into complex form or constructs by methods known from industrialapplications of textile manufacture.

The biopolymer is suited for extrusion and co-extrusion with differentcomponents, organic or inorganic in nature and polymeric or otherwise,including multiple components, multilayered types of fiber as well ashollow fibers and tubes.

Example 2 Forming the GD-Tube

Cohesive biopolymer gels obtained as described in Example 1 above weretransferred to a glass Petri dish and heated at 100° C. for 1-2 hrs in adry oven until ethanol was completely evaporated. Then the polymericsubstance was cooled to room temperature and transferred to a hot (100°C.) mold for preparation of sleeves or membranes by a compressionmolding procedure. After cooling the formed item was removed from themold and dried to a constant weight.

When NVR-7 was produced, it was found to be water insoluble and thus hasa longer retention time to biodegradation compared to NVR-3 and NVR-5.Incubation of NVR-7 matrix in sterile PBS at 37° C. for 4 month did notcause any changes in the construct appearance, integrity and/or weight.

Example 3 Testing the GD Tube to Serve as a Stent-Sleeve, or Coating

A GD-Tube in the length of 5 mm with a diameter of 2 mm was stretchedover a balloon carrying a coronary stent. The balloon was inflated to 16atmospheres with water. The sleeve remained intact under two inflationcycles of 16 atmospheres. This ability of the cohesive biopolymer forstretching displays its potential for serving as stent-sleeve to lowerrestenosis and thrombosis rates after angioplasty.

Example 4 Use of the GD Tube for Enclosing Neuronal Implant

The methodologies for the maintenance, growth and differentiation ofneuronal cultures are known to be most sophisticated. Therefore, anextracellular matrix (ECM) milieu that mimics the in vivo substrate andrequirements of neuronal cells is most desirable.

Tissue culture methods have gained attention as a substitute for the useof in vivo animal models. One direction was devoted to the creation andsimulation in vitro of the in vivo environment, nature and compositionof the extracellular matrix (ECM) for the cultured cells or explants. Asdisclosed previously by one of the present inventors (WO 02/39948), twomajor components, namely Hyaluronic acid (HA) and Laminin (LN), haveemerged as essential candidates specially for neuronal and glial cellcultures.

The combination of HA and LN into one viscous adhesive gel (HA-LN gel)has provided a biomatrix for growing neuronal cells and explants thatderived from both the central and the peripheral nervous systems. Thecombination of HA and LN, which are major components of the ECM havebeen introduced by the inventors as substrates for growing neuronalcells and explants derived from both the central and peripheral nervoussystems.

It was disclosed previously (WO 02/39948) that in addition to providinga useful substrate matrix for a broad range of cell types in vitro, theHA-LN gel serves as a highly advantageous biocompatible implant and asdelivery vehicle for transplantation.

Nevertheless, it turns out that in order to improve the mechanicalproperties it is desirable to enclose the HA-LN gels in a more rigidscaffold prior to implantation into a patient. This is achieved by useof the dextran sulfate-gelatin cohesive biopolymer of the presentinvention as a scaffold enclosing the HA-LN gel implant, the latter withour without cells.

The NVR guiding tube (denoted herein as GD tube) will be filled with theNVR-N-Gel with or without cells.

Example 5 Use of the GD Membrane for Closing a Tracheal Hole

Air leakage, structure collapse, flow obstructions, airways occlusions,vascular compression, hypotonicity, myoelasticity and respiratorydistress are characteristic symptoms in pediatricslaryngo-tracheo-bronchomalacia and/or stenosis.

Bronchomalacia is due to cartilaginous deficiency in the tracheal orbronchial wall, occurring in children under six months. There iscollapse of a mainstem bronchus, on expiration. There are two types ofBronchomalacia Primary bronchomalacia is due to a deficiency in thecartilaginous rings. Secondary bronchomalacia may occur by extrinsiccompression from an enlarged vessel, a vascular ring or a bronchogeniccyst. Both types may results in compressive lesions that may beidentified by MRI or CT examination. Bronchomalacia is alife-threatening illness in pediatric medicine.

Similar symptoms of Bronchomalacia may results from prolonged intubation(tube therapy), which can induce intra-tracheal scarring and fibrosis,leading to the above fatal pathologies.

NVR-7 membrane was examined as a treatment of deformed airways. Theexperiment was performed in vitro, in lung taken form a healthy rabbit,as well as in vivo, by deliberately forming a cut in the rabbit lungtrachea. After the cut was formed, an NVR-7 membrane of 1.0 cm×0.5 cmwas sewed to cover the cut. In both experiments the membrane cloggedcompletely the airway system, and enable a normal function of the lung.After the surgery, the rabbit was able to breath normally.

Example 5 Characterization of the GD Biopolymer

The GD biopolymer is produced as a coprecipitate of two simple polymericmolecules: dextran sulfate and gelatin. A series of tests were performedto compare the original raw materials and the new formed cohesivebiopolymer gel, as described herein below:

Gel Filtration Chromatography (GFC)

Gel filtration chromatography (GFC) profile of substances depends on themolecular weight as well as the 3-D shape of the molecule. Ten mg ofpolymeric substance dissolved in double distilled water was placed onthe column of Sepharose CL-6B and eluted with double distilled water.The excluded (void) volume was determined employing dextran blue of2×10⁶ Daltons, eluted in tube # 7 (11.5 ml) 1.5 ml/tube.

Polysaccharides of 1×10⁶ and up were excluded. Sugars were followed bythe phenol method for neutral sugars (1 ml sample+1 ml 5% phenolsolution+5 ml of concentrated sulfuric acid). The orange color developedwas read in a spectrophotometer at a wavelength of 490 μm. As shown inFIG. 4, the chromatogram of dextran sulfate alone (FIG. 4 a) and thechromatogram of the novel GD biopolymer (FIG. 4 c) are notablydifferent.

Similarly, gel filtration chromatography was performed to comparegelatin alone and the GD biopolymer.

Ten mg of polymeric substance dissolved in 0.5 ml of DMSO was placed onSepharose CL-6B column. Elution was performed with a 10% DMSO solution,collecting fractions of 1.5 ml each. Proteins having molecular weightsof 4×10⁶ Daltons and above were eluted at the void volume. Proteins weredetected by spectrophotometer at a wavelength of 280 nm. Again, thechromatogram profile of gelatin (FIG. 4 b) is distinctly different fromthe profile of the biopolymer (FIG. 4 c).

Nuclear Magnetic Resonance Spectroscopy (NMR)

FIG. 5 a-c describe NMR analyses of gelatin, dextran sulfate and thenovel GD biopolymer. The results clearly show that the new biopolymer isdistinguished from the original raw materials.

Infra Red Spectrometry

FIG. 6 shows the infrared spectra of gelatin and dextran sulfate as rawmaterials in comparison with the spectrum of the GD biopolymer.

A gross analysis of the spectrum, showing that the spectrum of the GDbiopolymer differ from those of the raw material molecules, suggests theformation of a new polymeric substance, as was shown by the results ofthe other tests described above.

Example 6 Characterization of GD Biopolymers with Different Degrees ofCross-Linking

The degradation rate of the polymer can be controlled by the extent andtype of the cross-linking between the polymer molecules. The method ofthe present invention, reacting the biopolymer with reducing sugars,resulted in intensive cross-linking of the biopolymer molecules.Comparing a full hand of cross-linking agents showed pentosemonosaccharide ribose as the best agent. The degree of cross-linking iscontrollable by the sugar concentration, temperature and the length ofthe reaction.

Comparing the GD properties before and after cross-linking examined theinfluence of the cross-linking degree on the properties of the GDbiopolymer.

Swelling Test

The membranes swelling studies were conducted using two media, namely,distilled water and simulated saliva solution. Each sample of membrane(NVR-3, surface area 40 mm²) was dried by vacuum for 4 h, weighed andplaced in a pre-weighed stainless steel wire mesh with sieve openings ofapproximately 200 μm. The mesh sieve with the film sample was submergedinto 25 ml medium placed in a plastic beaker. Increase in weight of themembranes was determined at successive time intervals until a constantweight was obtained. Each measurement was repeated three times. Thedegree of swelling was calculated using the following parameters:$\frac{W_{t} - W_{0}}{W_{0}} \times 100\%$

Where W_(t) is the weight of the membrane at time t; and W₀ is theweight of membrane at time zero.

The samples were tested before cross-linking, after cross-linking bydehydrothermal treatment and after cross-lining by a combination ofdehydrothermal treatment and sugars.

FIGS. 7 and 8 depict the degree of swelling of the GD membranes indistilled water and simulated saliva solution, respectively, before andafter cross-linking. The degree of swelling was higher in distilledwater compared to saliva solution. This finding suggests that ionicstrength and pH play an important role in affecting the swelling of themembranes. The rate of swelling for the GD membranes beforecross-linking was higher compare to membranes after thermalcross-linking and significantly lower for membranes after thecombination of thermal treatment with cross-linking by sugars. Thesedata indicate that both the thermal method and the sugars cross-linkingdecrease the rate of water uptake and hydration, as a result ofincreasing the degree of cross-linking.

In Vitro Biodegradation

Gelatin-dextran sulfate-membranes (GD membranes, NVR-3) were preparedand cross-linked by ribose. Samples, prepared for testing, were dried byvacuum for 4 h, weighed and fully immersed in the physiologicalsolution, supplemented with 20% fetal bovine serum at 37° C. for thespecified period of time. At each specified time period throughout theduration of the incubation time, the solution was replaced and sampleswere removed, dried and weighed. By measuring the weight change duringthe 30 days duration of the assay, the degradation rate was calculated.FIG. 9 shows that the ribose cross-linked preparations are degraded at arate of 2-2.5% per day.

Example 7 Porosity of the GD Biopolymer

The structure of a dry GD membrane (NVR-3) was examined by ScanningElectron Microscope (SEM), before and after incubation of the membranein neuronal cell culture medium for 24 days. As shown in FIG. 10, thedry membrane appears as a continuous dense solid with a randomly porousstructural; the size of the pores was 20-70 μm.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed chemical structures andfunctions may take a variety of alternative forms without departing fromthe invention.

REFERENCES

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1-42. (canceled)
 43. A biocompatible cohesive biopolymer gel comprisinga coprecipitate of at least one fibrillar protein and at least onesulfated polysaccharide.
 44. The biocompatible cohesive biopolymer gelof claim 43 wherein the coprecipitate is formed in the absence of anexogenous cross-linking agent in the presence of a volatile organicsolvent.
 45. The biocompatible cohesive biopolymer gel of claim 43wherein the coprecipitate is formed at a pH of at least 2 pH units aboveor below neutral pH.
 46. The biocompatible cohesive biopolymer gel ofclaim 45 wherein the coprecipitate is formed at an acidic pH, between pHof about 2.0 and pH of about 5.0.
 47. The biocompatible cohesivebiopolymer gel of claim 45 wherein the coprecipitate is formed at abasic pH between pH of about 9.0 and pH of about 12.0.
 48. Thebiocompatible cohesive biopolymer gel of claim 43 wherein the protein isselected from the group consisting of collagen, elastin, fibrin, albuminand gelatin.
 49. The biocompatible cohesive biopolymer gel of claim 48wherein the protein is gelatin.
 50. The biocompatible cohesivebiopolymer gel of claim 43 wherein the sulfated polysaccharide isselected from the group consisting of dextran sulfate, chondroitinsulfate, heparin, heparan sulfate, keratan sulfate, dermatan sulfate,algal sulfated polyglycan, or a synthetic sulfated polysaccharide. 51.The biocompatible cohesive biopolymer gel of claim 50 wherein thesulfated polysaccharide is dextran sulfate.
 52. The biocompatiblecohesive biopolymer gel of claim 51 wherein the dextran sulfate has amolecular weight in a range of from about 4,000 Dalton to about 500,000Daltons.
 53. The biocompatible cohesive biopolymer gel of claim 52wherein the dextran sulfate is selected from (a) a high molecular weightdextran sulfate having a molecular weight in a range of from about300,000 Dalton to about 500,000 Dalton; and (b) a low molecular polymerdextran sulfate having a molecular weight in a range of from about 5,000Dalton to about 10,000 Dalton.
 54. The biocompatible cohesive biopolymergel of claim 43 wherein the cohesive biopolymer comprises gelatin anddextran sulfate.
 55. The biocompatible cohesive biopolymer gel of claim54 comprising 30% to 70% of dextran sulfate.
 56. The biocompatiblecohesive biopolymer gel of claim 54 comprising 30% to 70% of gelatin.57. The biocompatible cohesive biopolymer gel of claim 54 comprisingabout 50% gelatin and about 50% dextran sulfate.
 58. The biocompatiblecohesive biopolymer gel of claim 43 further comprising at least onesubstance selected from anticoagulants, adhesive molecules, growthfactors, enzymes, antioxidants, antifibrotic substances, positivelycharged molecules, a peptide rich in positively charged amino acids, andnutritional elements.
 59. The biocompatible cohesive biopolymer gel ofclaim 44 further comprising bridges formed by subsequent addition of across-linking agent to the coprecipitate formed.
 60. The biocompatiblecohesive biopolymer gel of claim 59 wherein the cross-linking agent isselected from the group consisting of a monosaccharide: ribose, glucose,mannose and xylose; factor XIII; lysyloxidase; a carbodiimide; and anoxidizing agent.
 61. The biocompatible cohesive biopolymer gel of claim43 further comprising at least one bioactive compound selected from thegroup consisting of a hormone, a growth factor, a proteolytic enzyme, ananti-fibrotic agent, a coagulative agent, an extracellular matrixcomponent, an anti oxidant, a natural or synthetic polymer.
 62. Thebiocompatible cohesive biopolymer gel of claim 43 wherein thecoprecipitate is formed into fibers, sheets, sponges, fabrics or tubes.63. The biocompatible cohesive biopolymer gel of claim 43 wherein thecoprecipitate is formed as a coating for a stent, wherein the stent isselected from a vascular stent and a tracheal stent.
 64. Thebiocompatible cohesive biopolymer gel of claim 43 wherein thecoprecipitate is formed into a scaffold.
 65. The biocompatible cohesivebiopolymer gel of claim 64 wherein the scaffold encloses hyaluronicacid-laminin gel.
 66. The biocompatible cohesive biopolymer gel of claim43 wherein the coprecipitate is formed into a scaffold for enclosingneuronal cells.
 67. The biocompatible cohesive biopolymer gel of claim66 wherein the scaffold enclosing neuronal cells further encloseshyaluronic acid-laminin gel.
 68. The biocompatible cohesive biopolymergel of claim 43 formed into a scaffold for use as a cell bearingimplant.
 69. An implant comprising a biocompatible cohesive biopolymergel according to claim
 43. 70. A method for preparing a biocompatiblecohesive biopolymer gel suitable as an implant in a human or animal,which comprises: providing a solution of a fibrillar protein; providinga solution of sulfated polysaccharide; combining the two solutions at apH of at least 2 pH units above or below neutral pH in the absence of anexogenous cross-linking agent to form a coprecipitate of cohesive gel;and precipitating the cohesive gel with a volatile organic solvent. 71.The method of claim 70 wherein the fibrillar protein is gelatin.
 72. Themethod of claim 70 wherein the sulfated polysaccharide is dextransulfate.
 73. The method of claim 72 wherein the dextran sulfate has amolecular weight in a range of from about 4,000 Dalton to about 500,000Daltons.
 74. The method of claim 73 wherein the dextran sulfate isselected from (a) a high molecular weight dextran sulfate having amolecular weight in a range of from about 300,000 Dalton to about500,000 Dalton; and (b) a low molecular polymer dextran sulfate having amolecular weight in a range of from about 5,000 Dalton to about 10,000Dalton.
 75. The method of claim 70 wherein the pH is an acidic pHbetween pH of about 2.0 and pH of about 5.0.
 76. The method of claim 70wherein the pH is a basic pH between pH of about 9.0 and pH of about12.0.
 77. The method of claim 70 wherein the volatile organic solvent isan alcohol.
 78. The method of claim 70 further comprising forming thegel into fibers by a process selected from the group consisting of dryextrusion, gel extrusion, melt extrusion, solution extrusion, spinningextrusion, spraying of nanofibrils and combinations thereof.
 79. Themethod of claim 70 further comprising shaping the biocompatible cohesivebiopolymer gel.
 80. The method of claim 70 further comprisingincorporating a bioactive substance into the biopolymer.
 81. A kit forcarrying out extemporaneously a method according to claim 70, the kitcomprising at least one dose of each constituent solution necessary toobtain the coprecipitate which forms the biocompatible cohesivebiopolymer gel.
 82. A composition for sustained release of a bioactivesubstance comprising a bioactive substance within a biocompatiblecohesive biopolymer gel according to claim
 43. 83. A method forrepairing a lesion in a tissue, comprising applying the biocompatiblecohesive biopolymer gel of claim 43 at a site of the lesion.
 84. Themethod of claim 83 wherein the lesion is a tracheal lesion.